International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 Bacterial biofilm formation on urologic devices and heparin coating as preventive strategy Peter Tenke , Claus R. Riedl , Gwennan Ll. Jones , Gareth J. Williams , David Stickler , Elisabeth Nagy a Department of Urology, Jahn Ferenc South-Pest Hospital, H-1204 Budapest, Köves u. 2-4, Hungary b Department of Urology, Thermenklinikum Baden, Baden, Austria c Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK d Faculty of Medicine, Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary In the process of endourological development a variety of foreign bodies have been invented besides urinary catheters, on which biofilm can be formed. Bacteria in the biofilm are less susceptible to antibiotics. An additional problem of medical biomaterials in the urinary tractenvironment is the development of encrustation and consecutive obstruction. The most promising prevention strategy for bacterial biofilmsis the production of materials with anti-adhesive surfaces such as heparin. Although heparin-coated ureteral stents are expensive, they justifytheir cost. Our studies show that such devices are protected against incrustation and biofilm formation for a longer period of time: 6–12months, both in vitro and in vivo.
2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Keywords: Biofilm formation; Antimicrobial susceptibility; Incrustation; Obstruction; Heparin coating manent urinary catheters and stents are the most commonbiomaterial implants comparable to contact and intraocular It is known that bacterial biofilms can colonise the sur- lenses or hip and knee implants faces of both tissues and implanted medical devices. Theprocess of biofilm formation and the impact on the develop-ment and clinical course of infectious diseases, however, are 2. Mechanism of biofilm formation
still poorly understood. Effective preventive and therapeuticstrategies still need to be developed for device-associated The formation of biofilm generally consists of several infections. By definition, a biofilm is an accumulation of main steps: the first step is the deposition of the microorgan- microorganisms and their extracellular products forming a isms, next follows their attachment by microbial adhesion structured community on a surface.
and anchorage to the surface by exopolymer production. Af- It is evident that with the steadily increasing number of ter this process their growth, multiplication and dissemina- biomaterial devices used in urology for urinary drainage tion can be observed ( (catheters, ureteral and prostatic stents) as well as implants The initial event in this process is bacterial adhesion and for replacement of lost body functions (sphincters and other the deposition of a host urinary component on the surface continence devices, penile prosthesis), biofilm formation of the biomaterial leading to the formation of a condition- and device infection is an issue of growing importance. In ing film. This film consists of proteins, electrolytes and addition, new tissue surfaces are created by using bowel some unidentified molecules The types of compo- segments for partial or complete replacement of the lower nents that form the conditioning film depend on the surface urinary tract. According to a North American survey, per- characteristics (chemistry, charge and hydrophobicity).
Many of the protein molecules in the conditioning film ∗ Corresponding author. Tel.: +36-12847610; fax: +36-12856380.
play an active role in the bacterial adhesion process. The E-mail address: [email protected] (P. Tenke).
conditioning film does not cover the entire implant surface 0924-8579/$ – see front matter 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

P. Tenke et al. / International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 3. Antimicrobial susceptibility of biofilms
Bacteria within the biofilms differ both in behaviour and in phenotypic form from the planktonic, free-floatingbacteria. Conventional clinical microbiology can detectonly the planktonic, free-floating bacteria, which are ab-solutely different from bacteria enclosed in the biofilm The failure of antimicrobial agents to treat biofilms has been attributed to a variety of mechanisms In general, organisms encapsulated in the biofilm grow moreslowly than the planktonic organisms, probably because theencapsulated bacteria have a decreased nutrient and oxy-gen supply leading to a decreased metabolic rate and, asa consequence, to a decreased antimicrobial susceptibility.
This may lead to a less susceptible genotype selecting a re-sistant population. Furthermore, antimicrobial binding pro-teins are poorly expressed in these slow-growing biofilmbacteria.
The biofilm matrix itself often delays or impedes the dif- fusion of antibiotic molecules into the deeper layer of thefilm (extrinsic resistance).
Bacteria within the biofilm are phenotypically different Fig. 1. Formation of biofilm.
from their planktonic counterparts. Antimicrobial agentswould therefore frequently fail to eradicate them. Bacteria completely, but rather forms a "mesh-like" covering within a biofilm activate many genes, which change their Several factors are thought to influence bacterial adhe- surfaces and other molecular targets, reducing the suscep- sion to foreign body surfaces, such as biomaterial surface tibility to antimicrobial agents (intrinsic resistance). It is characteristics, bacterial surface features and the behaviour suggested that these phenotypic changes are more impor- of microorganisms and the presenting clinical condition tant for antimicrobial resistance than the external resistance mechanisms such as biofilm matrix or glycocalyx.
The biofilm is usually built up of three layers ( Bacteria within a biofilm can analyse the external en- The linking or conditioning film is attached to the surface vironment, develop interbacterial communication and may of a tissue or biomaterial, the biofilm base consisting of transfer genetic information and plasmids within biofilms.
microorganisms and the surface film acts as an outer layer As a consequence, bacteria in biofilms may survive the use where planktonic organisms can be released free-floating of antibacterial agents at concentrations 1000–1500 times and spread to the surrounding compartments The higher than needed to eradicate planktonic bacteria of the development of the biofilm is shown in same species.
Fig. 2. Composition of the biofilm.

P. Tenke et al. / International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 Fig. 3. Scanning electron microscope of a developing biofilm. Formation of the biofilm on the (a) outer surface of the polyurethane stent and (b) innersurface of the polyurethane stent.
4. Management of biofilm infection
ure of eradicating chronic bacterial infections with biofilms.
Antimicrobial treatment may be effective in "young" Urine cultures of planktonic bacteria and the definition of biofilms that developed within 24 h or less their antimicrobial susceptibility may contribute to the fail- Wollin et al. demonstrated that ciprofloxacin and ofloxacin

P. Tenke et al. / International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 care. The crystalline deposits can be hard and abrasive andcan traumatise the bladder mucosa and urethra. Obstructionof urinary flow through the catheter may cause either in-continence due to leakage of urine around the catheter orpainful distention of the bladder due to urinary retention.
Bacteriuria is always found in these patients, therefore re-tention and vesico-ureteral reflux may facilitate ascendinginfection of the urinary tract, culminating in episodes ofpyelonephritis, septicaemia and shock Thus, unde-tected catheter blockage may lead to life-threatening compli- Fig. 4. Effect of antibiotics (+) on biofilm progression (adapted from cations Several studies reported that up to 50%of patients undergoing long-term catheterisation will requireunscheduled catheter replacement because the flow of urine rapidly reached a high urinary concentration and were able has been blocked by crystalline deposits to penetrate into conditioning bacterial biofilms and onto the Current approaches in preventing catheter blockage by stent surface. Significantly greater amounts of both antibi- encrustation (i.e. replacement of the catheter, changing the otics were absorbed onto the biofilm than to the stent surface type or size of catheter, increasing fluid intake, administra- It was also demonstrated that ciprofloxacin concen- tion of cranberry juice or acidifying drugs and washing the trations on biofilms surrounding urinary stents were signif- bladder/catheter with acidic, antiseptic or saline solutions) icantly higher than ofloxacin concentrations are frequently ineffective Recurrent catheter blockage Other studies showed that ciprofloxacin and ofloxacin might gives patients the reputation as "blockers" ffec- prevent microbial adhesion and biofilm formation for a tive procedures to prevent encrustation are definitely needed.
short period of time Goto demonstrated that Since the significance of biofilm formation has been other drugs such as trimethoprim-sulphamethoxazole and appreciated as the main problem of all implants and bio- tobramycin were less potent against biofilms compared to material devices, modification of the biomaterial surface the fluoroquinolones According to Kumon, a com- was regarded the most promising prevention strategy for bination therapy with fluoroquinolones and macrolides or bacterial biofilms. A variety of techniques have been de- fosfomycin seems to be most effective against biofilm infec- signed for this purpose, including the controlled release tions Most researchers believe that antibiotics of antimicrobial agents or antiseptics (such as minocy- can only slow down the progress of biofilm formation by cline, rifampicin, gentamicin, nitrofurantoin) incorporated eliminating unprotected planktonic bacteria and stopping or in the device material, surface coatings with silver and reducing the metabolic activity of bacteria on the biofilm other metals, surface modifications to change or increase surface However, during an acute hydrophobicity or to create functional groups with intrinsic febrile phase of a biofilm infection, antimicrobial therapy is antimicrobial activity, and anti-adhesive surfaces such as reasonable and essential because the planktonic and not the heparin and phosphorylcolin biofilm bacteria are responsible for febrile reactions Heparin with its antithrombogenicity and its strong elec- tronegativity that repells cellular organisms is an excellentcandidate for an anti-adhesive stent coating. In 1987, Rug- 5. Biofilms in catheter-associated urinary tract
gieri et al. showed a 90% reduction of bacterial adhesion on urinary catheter surfaces by heparin coating brandt et al. demonstrated the reduction of stent encrustation An additional problem of medical biomaterials in the by heparin coating in an experimental setting urinary tract environment is the development of encrusta-tion and consecutive obstruction. When the drained urinarytract becomes infected by urease-producing bacteria such as 6. An in vitro examination of the ability of
Proteus mirabilis, the bacterial urease generates ammonia heparin-coated catheters to resist encrustation by
from urea and elevates the pH of the urine. In this alka- crystalline P. mirabilis biofilm
line environment, crystals of magnesium ammonium phos-phate (struvite) and calcium phosphate (hydroxyapatite) are The ability of three catheter types to resist encrusta- formed and trapped in the organic matrix surrounding the tion and blockage by crystal-generating urine cultures of cells. Progression of these encrustations eventually blocks P. mirabilis isolated from patients encrusted catheters was the catheter lumen Clinical experience and examined in a laboratory model of the catheterised blad- laboratory studies have shown that all types of catheters cur- der Catheters (14 French) were inserted aseptically rently available are vulnerable to blockage by crystalline through a section of silicone tubing. They were attached to P. mirabilis biofilms The complications result- a glass outlet at the base of the vessel into a 200 ml glass ing from catheter encrustation seriously compromise patient chamber maintained at 37 ◦C. The catheter balloon was

P. Tenke et al. / International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 inflated with water securing the catheter in position and der at 0.5 ml per minute. The models were operated until the sealing the outlet from the vessel "bladder". The catheter catheters were blocked with encrustation. Low vacuum scan- was then attached to a drainage tube and reservoir bag.
ning electron microscopy (REM) was performed to visually Sterile pooled human urine was supplied to the bladder via assess the extent of encrustation at catheter cross-sections 1, a peristaltic pump. Thus, a residual volume of about 30 ml 4, 10 and 30 cm from the tip.
was collected in the vessel below the level of the catheter Whereas the hydrogel-coated latex catheter was blocked eyehole. As urine was supplied to the model the overflow after an average of 28.1 h in four experiments, time un- drained through the catheter into the collecting-bag.
til blockage was significantly longer for the heparin-coated The three catheters tested were a latex catheter with hy- catheter (58.2 h) and silicone catheter (54.8 h) in the setting drophilic coating, a silicone catheter and a heparin-coated described. However, all three types of catheters were vul- silicone catheter. After inoculation of the sterilised urine nerable to Proteus blockage.
with the P. mirabilis strain, the organisms were allowed to es- Encrustation (REM) was observed only on hydrogel- tablish themselves in the model for 1 h. The peristaltic pump coated and silicone catheters especially around the eyeholes was then switched on and fresh urine supplied to the blad- and balloon but no encrustation was found on heparin-coated Fig. 5. (a) A heparin-coated ureteral stent which remains unaffected by biofilm formation and incrustation. (b) An uncoated ureteral stent with a biofilmformed on its inner surface.

P. Tenke et al. / International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 Fig. 6. Incrustation, biofilm formation and colour change of heparin-coated ureteral stents (Heparius) and uncoated polyurethane stents (PUR) within a6-week observation period. Y-axis is—0: no change, 1: moderate change and 2: significant change.
catheters; the blockage was caused by plugs of clear gel-like tomy tubes remained unaffected for the whole 6–8 weeks indwelling periods, whereas uncoated tubes got obstructedwithin 2–3 weeks.
This pilot study showed that no biofilms were de- 7. Clinical evaluation of encrustation and biofilm
tectable on heparin-coated stents whereas significant formation on heparin-coated ureteral stents and
biofilms were demonstrated in 33% of uncoated stents.
Mild incrustation was observed in 10% of heparin stentscompared to significant incrustation in 50% of uncoated In a pilot study the encrustation of heparin-coated stents, and incrustations/biofilms were demonstrable on ureteral stents was compared to uncoated polyurethane uncoated stents as early as 2 weeks after implantation stents. Twenty heparin-coated and 20 uncoated stents were inserted into obstructed ureters in a prospective randomisedstudy under sterile conditions and left indwelling for peri-ods between 2 and 6 weeks. The stents were then removed 8. Extended indwelling times for heparin-coated
under sterile conditions, sealed in sterile covers and sent for electron-microscopic evaluation. Nephrostomy tubeswere used in two patients with permanent bilateral external In 10 patients with permanent ureteral stent drainage, urinary drainage suffering from frequent encrustation ob- heparin-coated stents were left indwelling for 6–8 months struction of their silicone catheters that resulted in repeated (Group I). In all patients bacteriuria was demonstrable at the emergency visits. In these patients a heparin-coated and an time of heparin-coated stent insertion; from previous stents.
uncoated nephrostomy tube were used simultaneously for In three patients with uretero-enteral anastomosis stricture either side so that direct comparison of encrustation status in an ileal conduit, heparin-coated stents were left for 1 year was possible.
Electron microscopy showed a significant difference be- No obstruction/blockage of the stents was observed dur- tween heparin-coated and uncoated ureteral stents. ing this time in group I. On REM none or only minimal en- the two types of stents that react to biofilm crustations were found after this prolonged indwelling time.
formation in different ways. Two weeks after the insertion, In the difficult bacteria-exposed Group II situation, the two types of deposits could be detected on the surfaces of silicone stents were found to be obstructed after 7 weeks the uncoated stents—amorph anorganic deposits consisting while the hydrogel-coated stents in 5 months, whereas all of of mineralised crystals and another of bacterial biofilms.
the heparin-coated stents were unaffected after 12 months Heparin-coated stents were unaffected by encrustations. Af- of indwelling time.
ter 6 weeks of indwelling time, all uncoated stents showed Heparin-coated ureteral stents are more expensive than varying degrees and forms of deposits. Within the limited standard stents. However, with longer indwelling times and observation period of this pilot study none of the uncoated reduction of the number of stent exchange procedures, the stents became totally obstructed. The heparinized nephros- total costs for heparin stents should be reduced compared to

P. Tenke et al. / International Journal of Antimicrobial Agents 23S1 (2004) S67–S74 Fig. 7. Costs per year for heparin-coated ureteral stents (Heparius) and uncoated polyurethane stents (PUR).
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